Diagram of plasmid pBend2 with the φ29 sequence of region 1 and the mutated sequences Mut1, Mut2 and Mut3 cloned

<p><b>Copyright information:</b></p><p>Taken from "A precise DNA bend angle is essential for the function of the phage φ29 transcriptional regulator"</p><p>Nucleic Acids Research 2005;33(1):126-134.</p><p>Published online 07 Jan 2005</p><p>PMCID:PMC546135.</p><p>© 2005, the authors © </p> Top lane, enzymes organization on the polylinker of pBend2 around the cloned region 1, where the number corresponds to the restriction enzymes targets of the plasmid: BamHI (1), SspI (2), NruI (3), StuI (4), SmaI (5), PvuII (6), EcoRV (7), DraI (8), XhoI (9), SpeI (10), NheI (11) and BglII (12). Scheme of the 72 bp DNA fragments cloned, with the two inverted repeats indicated in boxes as sites 1 and 2. The nucleotide sequence of wild-type (pBend2-wt) region 1 and the modifications of the sequence on Mut1 (pBend2-Mut1), Mut2 (pBend2-Mut2) and Mut3 (pBend2-Mut3) are shown. It should be pointed out that Mut3 insert was opposite in orientation, relative to the other three constructions but here, to make clear its sequence characteristics, has been drawn anti-sense. The inverted repeats at the edges of sites 1 and 2 are denoted by horizontal arrows. Vertical arrow-heads denote the centre of the curvature induced upon p4 binding

Temperature dependency of the electrophoretical mobility of φ29 DNA fragments containing region 1 or 2

<p><b>Copyright information:</b></p><p>Taken from "A precise DNA bend angle is essential for the function of the phage φ29 transcriptional regulator"</p><p>Nucleic Acids Research 2005;33(1):126-134.</p><p>Published online 07 Jan 2005</p><p>PMCID:PMC546135.</p><p>© 2005, the authors © </p> Region 1 (R1) was a 200 bp PCR fragment with the sequence from position +98 of promoter A2c to position −6 of promoter A2b start sites. Region 2 (R2) was a 195 bp PCR fragment with the sequence from position +5 of promoter A2c to position −69 of promoter A2b start sites. Control (C) was a 198 bp fragment containing from positions +102 to −96 of early promoter C2 start site (). Lanes a–c and d–f, correspond to the native 4% acrylamide gel electrophoresis run at 65 and 4°C, respectively

Band-shift assay of the p4–DNA complex of the circularly permuted DNA fragments, which contain p4 binding region 1 wild-type (), Mut1 (), Mut2 () or Mut3 ()

<p><b>Copyright information:</b></p><p>Taken from "A precise DNA bend angle is essential for the function of the phage φ29 transcriptional regulator"</p><p>Nucleic Acids Research 2005;33(1):126-134.</p><p>Published online 07 Jan 2005</p><p>PMCID:PMC546135.</p><p>© 2005, the authors © </p> Protein p4 (160 nM) and purified circularly permuted DNA fragments from plasmids pBend2-wt, pBend2-Mut1, pBend2-Mut2 and pBend2-Mut3 were incubated for 15 min at 4°C and loaded directly onto the gel. Bands marked B denoted the p4–DNA complexes and bands labelled F, free DNA. The relative gel mobility of the p4 complexes was plotted against the position of restriction sites in the DNA fragment and expressed as the distance along the 5′–3′ (upper) strand from the left-hand BamHI site except in (D), where due to the orientation of the 72 bp insert, the distance was measured along the 5′–3′ lower strand from the BglII site. For the curve representations, data obtained from several experiments were used. Bending angles (α) are shown. Bend centre is shown as a vertical arrow. Gels show only some of the fragments analysed; other data are plotted in each figure

Table1_Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy.xlsx

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.</p

Table2_Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy.XLSX

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.</p

DataSheet1_Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy.pdf

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.</p